Halotolerant cyanobacterium Aphanothece halophytica contains NapA-type Na+/H+ antiporters with novel ion specificity that are involved in salt tolerance at alkaline pH

Abstract

Aphanothece halophytica is a halotolerant alkaliphilic cyanobacterium which can grow at NaCl concentrations up to 3.0 M and at pH values up to 11. The genome sequence revealed that the cyanobacterium Synechocystis sp. strain PCC 6803 contains five putative Na+/H+ antiporters, two of which are homologous to NhaP of Pseudomonas aeruginosa and three of which are homologous to NapA of Enterococcus hirae. The physiological and functional properties of NapA-type antiporters are largely unknown. One of NapA-type antiporters in Synechocystis sp. strain PCC 6803 has been proposed to be essential for the survival of this organism. In this study, we examined the isolation and characterization of the homologous gene in Aphanothece halophytica. Two genes encoding polypeptides of the same size, designated Ap-napA1-1 and Ap-napA1-2, were isolated. Ap-NapA1-1 exhibited a higher level of homology to the Synechocystis ortholog (Syn-NapA1) than Ap-NapA1-2 exhibited. Ap-NapA1-1, Ap-NapA1-2, and Syn-NapA1 complemented the salt-sensitive phenotypes of an Escherichia coli mutant and exhibited strongly pH-dependent Na+/H+ and Li+/H+ exchange activities (the highest activities were at alkaline pH), although the activities of Ap-NapA1-2 were significantly lower than the activities of the other polypeptides. Only one these polypeptides, Ap-NapA1-2, complemented a K+ uptake-deficient E. coli mutant and exhibited K+ uptake activity. Mutagenesis experiments suggested the importance of Glu129, Asp225, and Asp226 in the putative transmembrane segment and Glu142 in the loop region for the activity. Overexpression of Ap-NapA1-1 in the freshwater cyanobacterium Synechococcus sp. strain PCC 7942 enhanced the salt tolerance of cells, especially at alkaline pH. These findings indicate that A. halophytica has two NapA1-type antiporters which exhibit different ion specificities and play an important role in salt tolerance at alkaline pH.

FIG. 1.

Comparison of the deduced amino acid sequences of cation-proton antiporters. (A) Alignment of the deduced amino acid sequences of six cation-proton antiporters. The sequences were aligned with the program ClustalW. The amino acid residues conserved in all sequences are indicated by asterisks. Predicted membrane-spanning regions are indicated above the alignment. Site-directed mutated amino acid residues in Ap-NapA1-1 are enclosed in boxes. (B) Phylogenetic analysis of cation-proton antiporters. Multiple-sequence alignment and generation of the phylogenetic tree were performed with the ClustalW and TreeView software, respectively. The accession numbers for various antiporters are as follows: AB193603 for Ap-NapA1-1, AB193604 for Ap-NapA1-2, D64001 for Synechocystis sp. strain PCC 6803 Syn-NapA1 (slr0689), AF246294 for B. cereus GerN, U17283 for B. megaterium putative spore germination apparatus protein (GrmA), and M81961 for E. hirae NapA.

FIG. 2.

Effects of NaCl and LiCl on the growth of E. coli cells expressing Ap-NapA1-1, Ap-NapA1-2, and Syn-NapA1. (A) Immunoblot analyses of pApNapA1-1-, pApNapA1-2-, and pSynNapA1-expressing cells. Lane 1, pApNapA1-1-expressing cells; lane 2, pApNapA1-2-expressing cells; lane 3, pSynNapA1-expressing cells; lane 4, pTrcHis2C control cells. In each lane, 50 μg membrane proteins was loaded. Antiporter proteins were detected using an antibody raised against the His tag. (B) LB medium containing 30 mM KCl and different concentrations of NaCl. (C) LB medium containing 30 mM KCl and different concentrations of LiCl. The control TO114 cells and transformant TO114 cells expressing Ap-NapA1-1, Ap-NapA1-2, and Syn-NapA1 in the exponential phase were transferred to growth medium containing the different salts and pH. Each value is the average of three independent measurements of OD₆₂₀ at 9 h.

FIG. 3.

Cation-proton exchange activities measured by the acridine orange fluorescence quenching method. The control TO114 cells and transformed TO114 cells expressing Ap-NapA1-1, Ap-NapA1-2, and Syn-NapA1 were grown in LBK medium, from which everted membrane vesicles were prepared. The antiporter activity was measured as described in Materials and Methods. (A and B) Na⁺/H⁺ and Li⁺/H⁺ antiporter activities of Ap-NapA1-1, Ap-NapA1-2, and Syn-NapA1, respectively. (C and D) Na⁺/H⁺, K⁺/H⁺, Mg²⁺/H⁺ and Ca²⁺/H⁺ antiporter activities of Ap-NapA1-1 and Ap-NapA1-2, respectively. The final concentration of salts was 5 mM. Each value is the average of three independent measurements.

FIG. 4.

Comparison of Na⁺/H⁺ (A) and Li⁺/H⁺ (B) exchange activities of Ap-NapA1-1 and Ap-NhaP1. The experimental conditions were the same as those described in the legend to Fig. 3. Each value is the average of three independent measurements.

FIG. 5.

KCl dependence on growth and K ⁺ uptake in E. coli cells expressing Ap-NapA1-1, Ap-NapA1-2, and Syn-NapA1. (A) Growth of K⁺ uptake-deficient control LB650 cells and cells expressing pKT66 (positive control), Ap-NapA1-1, Ap-NapA1-2, and Syn-NapA1 in minimum medium containing different concentrations of KCl. (B) K ⁺ uptake of cells expressing pKT66. Ap-NapA1-1, Ap-NapA1-2, and Syn-NapA1 were detected with K⁺-depleted cells as described in Material and Methods. KCl (2 mM) was added at zero time. K ⁺ contents of the cells were measured as described in Materials and Methods. Each value is the average of three measurements.

FIG. 6.

Effects of K⁺ on Na⁺/H⁺ and Li⁺/H⁺ exchange activities. Everted membrane vesicles were prepared using TCDS buffer. For panels A, C, E, and G, the assay medium contained 140 mM choline chloride. For panels B, D, F, and H, KCl (140 mM) replaced choline chloride in the assay medium. (A, B, E, and F) Ap-NapA1-2-expressing cells; (C, D, G, and H) Ap-NapA1-1-expressing cells. (A to D) Na⁺/H⁺ exchange activity; (E to H) Li⁺/H⁺ exchange activity.

FIG. 7.

Schematic secondary structure model of Ap-NapA1-1. Acidic and basic amino acid residues in the loop regions are indicated by circled minus and plus signs, respectively. The conserved amino acid residues Gly140-Glu142 and Asp225-Asp226 are shown. E129 and K383 are charged amino acid residues not conserved among NapA antiporters.

FIG. 8.

Salt tolerance of Synechococcus sp. strain PCC 7942 cells expressing Ap-NapA1-1 and Ap-NhaP1. Freshwater Synechococcus sp. strain PCC 7942 cells transformed with the vector only (control), with Ap-NapA1-1, and with Ap-NhaP1 were grown in BG11 medium (A) or in BG11 medium containing 0.4 M NaCl (B) or 0.5 M NaCl (C) at pH 7.0. Each value is the average of three independent measurements.

FIG. 9.

Salt tolerance of Synechococcus sp. strain PCC 7942 cells expressing Ap-NapA1-1. Freshwater Synechococcus sp. strain PCC 7942 cells transformed with the vector only (control) and with Ap-NapA1-1 were grown in BG11 medium (open symbols) or in BG11 medium containing 0.3 M NaCl (solid symbols) at pH 7.0 (A) and pH 9.0 (B). Each value is the average of three independent measurements.